Stable poliomyelitis live virus vaccine



U id States Patent() STABLE POLIOMYELITIS LIVE VIRUS VACCINE Thisinvention relates to a free flowing granular vaccine comprisingnon-pathogenic poliomyelitis virus and gela- More-particularly, itrelates to said vaccine in a form administrable by theoral route.

Poliomyelitis virus has been adapted to grow andmade iionep'athogenicthrough the use of various known media such as chick embryo, rodentbrain, rodent spinal cord,

tissue culture, etc. Generally, it can be said thatthe activity of thevirus is stable under freezing conditions in the presence of moistureand that activity stability decreases as thetemperature is increased.At. about 40 C., for example, the about 3 days. At ordinaryrefrigerating temperatures (4 C.), the activity of the virus is lessstable than under freezing conditions. Moreover, although stable whenfrozen, the poliomyelitis Virus is essentially inactivated during afreeze-drying process.

There are several possible administrative routes which may be' followedwhen inducing immunity to poliomyelitis. The most logical andthe mostdesirable route to follow is the natural portal of entry, i.e., the oralroute,

employing an attenuated non-pathogenic living virus under quantitativelyand qualitatively controlled conditions. Until the advent of the presentinvention, there had been many obstacles to be overcome before thisultimately desired result could be obtained. One of thema'ny reasons Whythe oral route had not beensucces's-fully employed on a large scalebasis heretofore can be found in the above brief discussion of theinstability of the polio myelitis virus l I a In order to circumventthese difliculties, a number of proposals have been made prior to thisinvention; amongst others are a number of media for suspending thepoliomyelitis virus. Polyethylene glycol was used to suspend the virusand this combination'then filled intov gelatin capsules, It was'found,however, that this suspension medium Wasnot a suitable carrier, becausethe virus lost activity in this medium upon standing for about onemonth. An attempt was made to use glycerine, a well known pharmaceuticaland biological stabilizing medium; this attempt unsuccessful alsobecause it liquefied the gelatin shell when an attempt was made toencapsulate this virus suspension.

Live poliomyelitis vaccines have been administered orally in the past byadding a suspension of the poliomyelitis virus to milk after thawingfrom the frozen state. It has also been practiced in the past to addquantities of the virus as a suspension in polyethylene glycol tocapsules for oral administration. All of these methods requiredformulation immediately before administration because of the instabilityof the activity of the virus if allowed to stand in milk, polyethyleneglycol, other media, or in the unfrozen state. Many other proposals havebeen made; however, none have been successful in producing a vaccinesuitable for oral use whose activity was stable.

Moreover; it is most desirable to provide a c'ariier or poliomyelitisvirus usually dies in 2 V: suspending medium in which the activit of the'virusis stable and which is suitable for gelatin encap ulation,preferably a dry, free-flowing one. 5 Until the advent of the presentinvention, such a carrier ormediuni had not been found and the problemsof dispensing, distributing, storing, and administering apoliomyelitis-virusvaciiie had greatly hampered progress towardeffecting iii 'zation to the poliomyelitis virus.

7 It has now been found-that a suspension eras-yer the known types of thpoliou yelitis virus'inthc'ir pro gation media can be combinedwith nonhydrated g to produce a hydrated, free-flowing, encapsulablei ticulate,granular poliomyelitis tion is substantially dry in" appearance as isevidenced by its granular fre e-fljwingcharacteristics. This vacine'makes possible encapsulation of the poliomyelitisjivirus and storage oftheencapsulated vaccine for an extended period of time without adverselyaffecting the activitystability thereof. It is thus possible to producean oral poliomyelitis vaccine in large quantities which need not be madeup immediately at the time of administration. The virus activity of thisencapsulated poliomyelitis vaccine is stable at both room temperature(i.e.,.about 20 C.) and at ordinary refrigeration temperature (i.e.,about 4 C.) for periods of several months.

In formulating the vaccine of the present invention, a propagationmedium containing an attenuated p o1iomyelitis virus .is blended withthe gelatin by agitation. During agitation, the gelatin becomes hydratedwith the virus co'ntaining propagation medium and the virus is absorbedby the gelatin. During hydration, the vaccine evolves from an initialthin paste to a vaccine consisting ofdiscretc particles when hydrationis complete. The

vaccine thus produced is a free-flowing,granularjone whichcontains,throughouta substantiallyunifonn distribution of the virus.

Any known virus-propagation media may be used .for the purposes of thisinvention. Examples of-tl eselare chick. embryo, rodent, brain andspinal cord (eg'. those of the hamster, cotton rat, mouse, rat, etcJ,tissue culture, etc. The virus is propagated infany of theknown media ina nonathogenic form, and the resulting suspension of the virus in water,along with the tissue, salts, antibiotics, anti-fungal agents, etc.contained ,thereiu,.are collected and compounded with the gelatin asdescribed above. The attenuated non-pathogenic poliomyelitis virus usedin this invention maytiter from 10 to 10 doses of virus per cc. betweenabout 10 and 10 The titer of the preferred oral virus dose rangesbetween about 10 and 1 0 {Hie poliomyelitis virus, after propagation inthe tissue culture medium of the followingexamples (virus-medium No.1),usually titers about 10 to 10 the preferred tite'r for oral dosage beingobtained =by;dilution. After propagation in the rodent brain and spinalcord medium of the following examples (virus medium No. 2), thepoliomyelitis virus usually titers between about 10 and 10. Gelatin witha particle size of between about 10510 140 mesh (U.S. standardsieve'size) may be used for the purposes of this invention. Gelatinhaving a particle size of from about 60 mesh to 100 mesh is preferred.

. a. a -3 s vac;cine, the-virus activity of which remains stable over Inpractice, it will .generally ztiter .tional apparatus and procedures.

added; Examples of anti-bacterial agents which have been used arepenicillin, tetracycline, chlortetracyline,

streptomycimetc. These antibacterial and anti-fungal agents are absorbedalong with the water, virus, etc. contained in the virus-containingmedia during hydra- ,tion of the gelatin. The resulting free-flowinggranular vaccine may be encapsulated in either soft shell gelatincapsules or hard shell gelatin capsules utilizing conven- Various knownexcipients may be added prior to encapsulation to further enhance thefree-flowing characteristic of the vaccine of the present invention. Forexample, starches such as potato starch, milo starch and corn starchhave been utilized with the present invention in order to betterfacilitate encapsulation in soft shell capsules.

In general, the respective amounts of gelatin and virus suspension mayvary over a wide range; for example, it has been found possible tosuccessfully formulate the vaccine of the present invention by utilizing10 parts ,by weight of gelatin to 1 part by volume of the virussuspension (25% to 75% by volume glycerine, preferably about 50%). Atthe other extreme, it has been found possible to formulate the vaccineof this invention successfully with 1 part by weight of gelatin to 4parts by volume of the virus suspension. If the amount of gelatin isincreased substantially beyond the 10:1 ratio described above,non-uniform distribution of the virus on the gelatin particles results,as there is insufficient liquid to initially completely wet down thesurfaces of all of the gelatin particles. At the other extreme, if theamount of gelatin is decreased beyond the 1:4 ratio described above, thecomposition becomes lumpy and wet and is thus handled with difliculty atbest. The preferred range for all three types of poliomyelitis virus isfrom ,1 to 2 parts by weight of gelatin to 3 parts by weight of thevirus suspension. If an excipient is added to this composition, it maybe added in an amount in the range of from about 5% to 20% by'weight ofthe excipient to the total weight of the gelatin and virus suspension;it

'has been found preferable to operate in a range from "about to 12% byweight.

There are several essential features of the new preparative method ofthis invention. First of all, in order to better facilitateencapsulation, the particle size of the 'gelatin starting materialshould fall within the range of about 10 to 140 mesh (U.S. standardsieve size). For

'best results, a gelatin particle size of from about 60 to 100 mesh ispreferred. Secondly, the temperature of all ingredients must becontrolled within a fairly narrow range of from about 5 C. to about 20C., and working conditions dictate a temperature from about 3 C. toabout 5 C. for optimum results. Finally, it is preferable that thegelatin be added in small quantities with agitation to thevirus-containing medium (i.e. by sponging). It is possible to add themedium to the gelatin.

-However, if so performed, it is necessary to exercise caution by slowaddition and rapid agitation in order to avoid extremely rapid hydrationwhich results in the production of large aggregates of gelatin which areincompletely hydrated.

If the above-described temperature limitations are not utilized, anunsatisfactory product is produced. If one exceeds the upper temperaturelimitation (20 0.), large, tacky, incompletely hydrated particles ofgelatin result from too rapid hydration. At these higher temperatures,

these large particles which areunsuitable for encapsulation will exceed,in terms of weight, the finer particles suitable for encapsulation. Infact, it is only when one uses temperatures of 10 C. and below that onecan be assured of completely satisfactory results. Below the lowertemperature limitation (5 C.), virus-containing solids' separate fromthe liquid phase. It is, therefore, preferable to operate above 0 C. Ifgelatin having a particle size outside the range of about 10 to 140 meshis utilized, it is unsuitable for encapsulationafter it has beenhydrated.

The following examples are given to more particularly illustrate thespecific detailsv of the present invention. Equivalent procedures andquantities will occur to those skilled in the art (e.g. chick embryo maybe used in propagating the non-pathogenic attenuated poliomyelitis virususeful for the present invention). The following examples are,therefore, not meant-to define the limits of the present invention,these being delmed only by the scope of the appended claims.

EXAMPLE 1 The following is a description of (1) a typical preparation ofa tissue culture medium and its use in growing Type I (SM strain), TypeH (TN strain), or Type III (Fox strain) poliomyelitis virus and (2) atypical preparation of mouse brain and spinal cord medium and its use inpreparing attenuated poliomyelitis virus of the same types and strainsas set forth in (1) above. These media will be referred to in theexamples that follow as virus-containing medium No. 1 andvirus-containing medium No. 2, respectively:

(1) Virus-containing medium No. 1

A. Growth of monkey kidney cells (see volume 2, Virology, August, 1956,pages 575 and 576).-Kidneys removed from Rhesus or Cynomolgus monkeysare chopped into small particles and digested overnight (about 15 hours)at 4 C. with 0.25% solution of trypsin and viable cells are recoveredtherefrom. Approximately 150,000 of these cells per cc. are suspended ina tissue culture medium consisting of:

Bovine serum ultra filtrate 15 volumes. Hank balanced 'salt solution 80volumes. Beef embryo extract 5 volumes. Penicillin units per milliliterof fluid.

Streptomycin 50 micrograms per milliter of fluid.

Hank balanced salt solution:

NaCl 8.0 g.

KCl 0.4 g.

CaCl (anhydrous) 0.2 g.

Mg.SO.,.7H O 0.2g. per 1000 ml.

Na HPO .2H O 0.06 g. of water.

KH PQ, 0.06 g.

Glucose 1.00 g.

NaHCO 0.35 g.

bottom of each container.

B. Growth 0 the virus.-At the end of the sixth day, the fluid content ofthe tissue culture medium is removed from each bottle and replaced with500 milliliters of tissue culture Medium No. 199 (see Reagents and Mediafor Tissue Culture and Virus Propagation, published by Difco). Thecontents of each bottle is then seeded with 5 milliliters of attenuatedpoliomyelitis virus (see volume 162 of the Journal of the AmericanMedical Association, December 1, 1956, page 1281). The seeded medium andcells are then incubated at 37 C. When substantially all of the cellsare destroyed (i.e., they no longer adhere Swiss Albino mice are eachinoculated with 0.03 milliliter of attenuated virus by theintra-cerebral of intraspinal route. When the mice become paralyzed, thebrains and spinal cords of each are harvested. A 20% weight/ volumesuspension is then prepared of the harvested brains and cords in normalsaline solution to which is added 50 micrograms of streptomycin permilliliter of fluid and 50 units of penicillin per milliliter of fluid.This is virus-containing medium No. 2, and like virus-containing mediumNo. 1 described above, it may then be compounded with gelatin or it maybe frozen and stored until it is desired to use it.

EXAMPLE 2 A Type I (SM strain) poliomyelitis virus vaccine wasformulated containing the following components:

Gelatin ('60-80 mesh, U.S. standard sieve size) gms 50 Corn starch gms13 Virus-containing medium No. 1 cc 37.5 Glycerine 37.5

TITER OF VACCINE ACTIVITY Times in Days Temp. Initial 10 10- 10-4.: 10-45 1mm 0 m 104.3 5-3 EXAMPLE 3 A Type I (SM strain) poliomyelitis virusvaccine was formulated following the procedure of Example 2 with thefollowing quantities of materials:

Gelatin (60-80 mesh) 23.8 gm., 35% w./w. Corn starch 6.8 gm., 10% W./W.Virus suspension (virus-containing medium No. 2-50% v./v. and

glycerine U.S.P.--50% v./v. 34 cc., 55% w./w.

All ingredients and utensils, except the corn starch, were chilled to 4C. before blending. The gelatin was hydrated slowly with agitation atlow temperatures as in Example 2. When hydration was complete, the cornstarch was added With agitation in three successive portions. Afree-flowing, particulate, granular vaccine of effective titer as inExample 2 was thereby obtained.

EXAMPLE 4 A Type II (TN strain) poliomyetlitis virus vaccine ofeffective titer and free-flowing, particulate, granular form wasformulated followin the" procedure and utilizing t he followingmaterials:

bf Example 2 A hydration temperature Otf 4 C. was utilized and whenhydration was complete corn starch was added in three successiveportions with agitation.

EXAMPLE 5 A Type II (TN strain) poliomyelitis virus vaccine of effectivetiter and free-flowing, particulate, granular form was formulatedfollowing the procedure of Ex ample 4 utilizing the followingingredients:

Gelatin (60-100 mesh) 'gm 70 Corn starch U.S.P. am 20 Virus-containingmedium No. 1 cc 5O Glycerine U.S.P. ..cc 50 At the completion of thecold sponging step (4 C.), the corn starch was blended until discreteparticles were formed, i.e., all aggregates broken.

EXAMPLE 6 A Type III (Fox strain) poliomyelitis virus vaccine ofeifective titer was formulated following the procedure and utilizing thesame ingredients (other than the virus itself), i.e. gelatin, starch,glycerine, and medium No. l, in the same quantities as Example 2. Ahydrated, freeflowing, particulate, granular vaccine was obtained which,when encapsulated in soft shell capsules, illustrated potencies bytissue culture titration similar to those found in Example 2.

EXAMPLE 7 comprising an aqueous attenuated non-pathogenic poliomyelitisvirus suspension adsorbed by substantially nonhydrated gelatin particlesfrom 10 to mesh as water of hydration, the amount of gelatin beingsufficient so that after hydration with the virus suspension, theparticles are substantially dry, said virus vaccine being freeflowing,particulate, granular, and storage-stable with respect to its activity.

2. The vaccine of claim 1 further characterized in that it contains ananti-microorganism agent.

3. The vaccine of claim 1 further characterized in I that it contains anexcipient.

4. A process of preparing a stable, orally administra ble free-flowing,particulate, granular poliomyelitis vaccine suitable for encapsulationwhich comprises mixing with agitation an aqueous suspension of anattenuated non-pathogenic poliomyelitis virus and substantiallynonhydrated gelatin having a particle size within the range of fromabout 10 mesh to about 140 mesh, at a temperature from about 5 C. toabout 20 C., the relative amounts of virus suspension and gelatinparticles being such that the gelatin particles after hydration by thesus pension are substantially dry.

(References on following page) 7 8 References Cited in the file of thispatent Salk: J.A.M.A., vol. 162, No. 16, Dec. 15, 1956 pp.

UNITED STATES PATENTS 4 2,166,074 Reicpel Ju1y 1 1, 1939 i";; 6 anFOREIGN PATENTS 6 Terzin: Proc. 'Soc. Exp. Biol. and Med., vol. 84, N).

738,454 Great Brit-aid Oct. 12, 1955 P- I 31 1955 1 30 N 3 ru Ia ew an.OTHER REFERENCES 6 s PP Sabin: J.A.M.A., vol. 162, No. 18, Dec. 29,1956, m Mfg. Chemist, February 1956, vol. 27, No. -2, pp.

pp. 1589-1596. R

1. AN ORALLY ADMINISTRABLE POLIOMYELITIS VIRUS VACCINE COMPRISING ANAQUEOUS ATTENUATED NON-PATHOGENIC POLIOMYELITIUS VIRUS SUSPENSIONADSORBED BY SUBSTANTIALLY NONHYDRATED GELATIN PARTICLES FROM 10 TO 140MESH AS WATER OF HYDRATION, THE AMOUNT OF GELATIN BEING SUFFICIENT SOTHAT AFTER HYDRATION WITH THE VIRUS SUSPENSION, THE PARTICLES ARESUBSTANTIALLY DRY, SAID VIRUS VACCINE BEING FREEFLOWING, PARTICULATE,GRANULAR, AND STORAGE-STABLE WITH RESPECT TO ITS ACTIVITY.